Spectroscopic Study of the Cobalamin-Dependent Methionine Synthase in the Activation Conformation: Effects of the Y1139 Residue and S-Adenosylmethionine on the B12 Cofactor

详细信息    查看全文

• 作者：Matthew D. Liptak ; Supratim Datta ; Rowena G. Matthews ; Thomas C. Brunold
• 刊名：Journal of the American Chemical Society
• 出版年：2008
• 出版时间：December 3, 2008
• 年：2008
• 卷：130
• 期：48
• 页码：16374-16381
• 全文大小：271K
• 年卷期：v.130,no.48(December 3, 2008)
• ISSN：1520-5126

文摘

The cobalamin-dependent methionine synthase (MetH) from Escherichia coli is a modular enzyme that catalyzes a methyl group transfer from methyltetrahydrofolate to homocysteine via a methylcob(III)alamin (MeCbl) intermediate, generating tetrahydrofolate and methionine (Met). Once every 2000 turnovers, the cobalamin cofactor is converted to the inactive cob(II)alamin (Co²⁺Cbl) form, from which MeCbl has to be recovered for MetH to re-enter the catalytic cycle. A particularly puzzling aspect of this reactivation process is that it requires the reduction of the Co²⁺Cbl species to cob(I)alamin (Co¹⁺Cbl) by flavodoxin, a reaction that would appear to be endergonic on the basis of the corresponding reduction potentials. To explore how MetH may overcome this apparent thermodynamic challenge, we have prepared the I690C/G743C variant of a C-terminal fragment of MetH (MetH^CT) to lock the enzyme into the activation conformation without perturbing any of the residues in the vicinity of the active site. A detailed spectroscopic characterization of this species and the I690C/G743C/Y1139F MetH^CT triple mutant reveals that the strategy employed by MetH to activate Co²⁺Cbl for Co²⁺ → Co¹⁺ reduction likely involves (i) an axial ligand switch to generate a five-coordinate species with an axially coordinated water molecule and (ii) a significant lengthening, or perhaps complete rupture, of the Co−OH₂ bond of the cofactor, thereby causing a large stabilization of the Co 3d_z²-based “redox-active” molecular orbital. The lengthening of the Co−OH₂ bond is mediated by the Y1139 active-site residue and becomes much more dramatic when the S-adenosylmethionine substrate is present in the enzyme active site. This substrate requirement provides MetH a means to suppress deleterious side reactions involving the transiently formed Co¹⁺Cbl “supernucleophile”.